Malaria Immunology and Pathogenesis Goals are:1) To understand how Malian children with hemoglobin S, alpha-thalassemia, G6PD deficiency, and ABO polymorphisms are protected from Plasmodium falciparum malaria; 2) To develop a profile of the acquisition of malaria immunity in Malian children;3) To examine aspects of pathogenesis due to malaria in Malian children and adults;4) To characterize the pharmacokinetics of artemisinin (ART) and the mechanisms of parasite clearance after drug treatment. To initiate these investigations, we conducted a 4-year longitudinal study of 1500 children ranging in age from 6 months to 18 years living in 3 villages in Mali (the Kenieroba study). One major effort is to determine the relative protection against malaria conferred by different red blood cell (RBC) polymorphisms, and all the enrolled children have been typed for a series of these polymorphisms. After diagnosis and treatment of 4207 episodes of malaria, we have developed a very detailed profile of age-dependent acquisition of resistance to malaria in this population. We have shown that sickle cell trait (HbAS) confers greater protection against malaria than other RBC polymorphisms. We are continuing to investigate the mechanistic basis for this protection:1) We identified a sub-cohort of children, selecting those with sickle cell trait (HbAS) and pairing them with age-matched HbAA controls, and we obtained plasma and cell samples for 3 years from this group. We have found that antibodies to a variety of merozoite proteins are lower in the HbAS children so that this is not the basis for their resistance;2) We developed a plasma reactivity assay using flow cytometry to measure antibody binding to infected red cells and showed that HbAS children do not display increased reactivity to the red cell surface; 3) In 2014 we have extended this work in two collaborative studies. We are evaluating the ability of sera from these children to limit parasitemia using an antibody-dependent respiratory burst assay (ADRB) and also a parasite opsonization assay. Results from both these assays are being correlated with protection from malaria infection in the children. Some children with malaria display severe pathology, but it has been unclear as to whether certain P. falciparum strains elicit more pathology or whether aspects of the host genetics or responses contribute to susceptibility. We are conducting a series of studies related to pathology during malaria including: 1) Reactivity of sera from adult males and females from Mali to various domains of a particular PfEMP1 protein, VAR2CSA, which has been implicated in pregnancy associated malaria. We have explored a major host effector function opsonization of parasitized red cells expressing VAR2CSA on the surface. We have completed and published this work in 2014 using reversal of opsonization as a novel application of the assay. Using this assay we have identified several domains within the VAR2CSA protein which are particular targets of opsonizing antibodies in multigravida Malian women; 2) We have continued to assess the in vitro and in vivo responses of Malian children to artesunate (ART), an effort prompted by reports from Asia of slow clearance of P. falciparum infections. We have not yet found this resistant phenotype but we are continuing to study the mechanisms by which parasites are cleared from the blood after treatment with ART and to investigate the role of anti-parasite antibodies in this clearance. Malaria Transmission Transmission of malaria is a critical aspect of the parasite life cycle but is poorly understood, so that we have increased our efforts to investigate parasite sexual stages and transmission in the field. In May of 2014 we completed a clinical protocol studying 500 individuals of all ages in Kenieroba. Volunteers were finger-pricked twice per month for a year and sera were collected by venipuncture three times during the year. Fingerprick samples collected will allow analysis of both DNA and RNA. This year we have developed protocols for analysis of parasite DNA by PCR directly on filter paper and we are applying these to all the samples. In addition, we have established methods for quantitation of gametocyte mRNA encoding Pfs25 using RT-PCR with the RNA samples. Thus we can identify both carriers of asexual and sexual stage parasites. These profiles will be compared throughout the year and correlated with age and area of residence. Selected serum samples will be tested for the capacity to inhibit transmission in membrane feeding assays to determine whether those living in endemic areas develop antibodies that can block transmission. Strategies to impact malaria transmission will be accelerated by a better understanding of those individuals primarily responsible for transmission to mosquito vectors. P. falciparum and evasion of mosquito immune system We had obtained experimental evidence that some African strains of P. falciparum can evade the mosquito immune system resulting in more effective transmission. Using a combination of QTL mapping and linkage group selection, we identified Pfs47 as the malaria gene that allows the parasite to infect mosquitoes without being selected for destruction. In Africa the Pfs47 gene is highly polymorphic. We are testing the general hypothesis that Pfs47 has to interact with a mosquito gene to disrupt the immune response and be transmitted more effectively, and that different alleles of Pfs47 work better to evade the immune system of certain mosquito vectors. If this hypothesis is correct, we expect that the different mosquito vectors in Africa will preferentially transmit parasites with certain alleles of Pfs47. The team in Mali is collecting mosquitoes from homes using the spray-catch method and doing ELISA assays to identify P. falciparum infected mosquitoes. Material from infected mosquitoes has been sent to NIH and we are extracting the genomic DNA, sequencing the Pfs47 gene and doing molecular genotyping of the mosquito vector. We received a total of 212 mosquito samples sent from Mali, collected during the 2010-2011 season. We sequenced the Pfs47 D2 domain (the most variable region) in 150 samples, and found that 111 mosquitoes had single P. falciparum infections, while 39 were infected with more than one parasite. We identified 11 haplotypes (based on the D2 domain sequence) in mosquitoes with a single infection and two of them were the most frequent, representing 55 and 18% of the samples. Regarding the temporal distribution of the 11 haplotypes, the largest diversity of haplotypes was found in the month of October. With our current sample size, we did not find a significant difference in the frequency of Pfs47 haplotypes between M-form and S-form A. gambiae mosquitoes. Malaria Vectors Previously our main efforts focused on (1) long distance migration of mosquitoes as means of re-colonization of Sahelian villages after population extinction, and (2) finding hidden shelters of African malaria mosquitoes during the Sahelian dry season. This work has expanded to become an independent project in 2014. We have also assisted Dr. Patrick Duffy and his LMIV associates who are working on clinical trials of a transmission blocking vaccine candidate. These studies require an insectary and mosquito feeding expertise in order to evaluate the functionality of antibody responses to the vaccine and we have supported Dr. Duffy and LMIV in this work. Sand fly / Leishmaniasis Program The program on leishmaniasis and its sandfly vectors is designed to describe the unexamined epidemiology of cutaneous leishmaniasis (CL) in Mali. Previously we showed that Leishmania major, the causative agent of cutaneous leishmaniasis, was the only parasite species found in sandflies, and that Phlebotomus duboscqi is the principal vector.